The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

Mentions:
Analysis of ultrathin sections revealed a significant decrease in number and density of synaptic vesicles in nerve terminals from SPLfl/fl/Nesmice compared to controls (Fig. 2a). Density of synaptic vesicles (SVs) localized in the area 300 nm from the border of the active zone was decreased in SPLfl/fl/Nesmice compared to controls (controls: 340.3 ± 12.3; SPLfl/fl/Nesmice: 129.1 ± 7.3 N/μm2; P < 0.0001) (Fig. 2b). The reduction was also evident in each of 100 nm steps from the border of the active zone, indicating that the number of SVs is reduced not only in the reserve pool, but also in the pool proximal to the active zone. Unexpectedly, the number of vesicles associated with the presynaptic membrane per μm2 of the active zone at rest was not reduced significantly (controls: 115.4 ± 48.2 and SPLfl/fl/Nesmice: 98.5 ± 38.0; P = 0.345; n = 28 synapses; two-tailed t-test). This result suggests that once available SVs can be efficiently recruited to sites of release. Further, the diameter of SVs in SPLfl/fl/Nesmice was significantly larger compared to controls (Fig. 2c). Similar findings were observed for mossy fiber synapses in CA3 region (suppl. Fig. 2). The number of clathrin-coated endocytic intermediates was significantly increased in nerve terminals with ablated SPL as compared to controls (controls: 0.13 ± 0.09 and SPLfl/fl/Nesmice: 1.77 ± 0.35 and; P < 0.001; n = 62 synapses; two-tailed t-test). An increased expression of dynamin was also observed in SPLfl/fl/Nesmice (Fig. 2d). No significant difference in the length of the active zone was found in synapses from SPLfl/fl/Nes and controls (0.23 ± 0.01 and 0.24 ± 0.01, P = 0.224; n = 62; two-tailed t-test).

Mentions:
Analysis of ultrathin sections revealed a significant decrease in number and density of synaptic vesicles in nerve terminals from SPLfl/fl/Nesmice compared to controls (Fig. 2a). Density of synaptic vesicles (SVs) localized in the area 300 nm from the border of the active zone was decreased in SPLfl/fl/Nesmice compared to controls (controls: 340.3 ± 12.3; SPLfl/fl/Nesmice: 129.1 ± 7.3 N/μm2; P < 0.0001) (Fig. 2b). The reduction was also evident in each of 100 nm steps from the border of the active zone, indicating that the number of SVs is reduced not only in the reserve pool, but also in the pool proximal to the active zone. Unexpectedly, the number of vesicles associated with the presynaptic membrane per μm2 of the active zone at rest was not reduced significantly (controls: 115.4 ± 48.2 and SPLfl/fl/Nesmice: 98.5 ± 38.0; P = 0.345; n = 28 synapses; two-tailed t-test). This result suggests that once available SVs can be efficiently recruited to sites of release. Further, the diameter of SVs in SPLfl/fl/Nesmice was significantly larger compared to controls (Fig. 2c). Similar findings were observed for mossy fiber synapses in CA3 region (suppl. Fig. 2). The number of clathrin-coated endocytic intermediates was significantly increased in nerve terminals with ablated SPL as compared to controls (controls: 0.13 ± 0.09 and SPLfl/fl/Nesmice: 1.77 ± 0.35 and; P < 0.001; n = 62 synapses; two-tailed t-test). An increased expression of dynamin was also observed in SPLfl/fl/Nesmice (Fig. 2d). No significant difference in the length of the active zone was found in synapses from SPLfl/fl/Nes and controls (0.23 ± 0.01 and 0.24 ± 0.01, P = 0.224; n = 62; two-tailed t-test).

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.